EP0080166B1 - Isolation d'antibiotiques chimiquement instables de solutions de fermentation - Google Patents

Isolation d'antibiotiques chimiquement instables de solutions de fermentation Download PDF

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Publication number
EP0080166B1
EP0080166B1 EP82110641A EP82110641A EP0080166B1 EP 0080166 B1 EP0080166 B1 EP 0080166B1 EP 82110641 A EP82110641 A EP 82110641A EP 82110641 A EP82110641 A EP 82110641A EP 0080166 B1 EP0080166 B1 EP 0080166B1
Authority
EP
European Patent Office
Prior art keywords
fermentation solution
resin
fermentation
antibiotics
activity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired
Application number
EP82110641A
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German (de)
English (en)
Other versions
EP0080166A1 (fr
Inventor
Gerhard Dr. Huber
Peter Dr. Schindler
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hoechst AG
Original Assignee
Hoechst AG
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Filing date
Publication date
Application filed by Hoechst AG filed Critical Hoechst AG
Priority to AT82110641T priority Critical patent/ATE15227T1/de
Publication of EP0080166A1 publication Critical patent/EP0080166A1/fr
Application granted granted Critical
Publication of EP0080166B1 publication Critical patent/EP0080166B1/fr
Expired legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
    • C12P17/184—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system containing a beta-lactam ring, e.g. thienamycin

Definitions

  • Streptomyces strains formed ⁇ -lactam antibiotics with carbapenem structures e.g. B. the thienamycin and olivanic acid group are characterized by particularly high antimicrobial activity and a broad spectrum of activity.
  • the antibiotics in this group are highly effective inhibitors or inactivators of ⁇ -lactamases of various origins. The isolation of these substances from the fermentation solutions is extremely difficult due to their extreme chemical instability.
  • the half-lives of chemical degradation in aqueous solutions in the medium pH range (pH 6-8) for thienamycin are between 0.3 and 6 hours depending on the conditions (J. Antibiot. 32, 1-12, 1979), for olivanic acids between 4 and 27 hours (J. Antibiot.
  • the adsorption can e.g. B. done so that one adds an antibiotic activity binding anion exchange resin to the fermentation solution and cools the fermentation solution quickly, for example by dilution with cold water to about 1-10, preferably 1-6 ° C.
  • Suitable anion exchange resins in the present case are preferably those with medium to strong basicity and with little crosslinking and porous structure such as.
  • the exchanger can be separated from the fermentation solution and mycelium by sedimentation (sedimentation) from the fermentation solution diluted as above.
  • the diluted fermentation solution containing the ion exchanger and mycelium is passed through a suitable sieve, e.g. B. a vibrating sieve (z. B. ®Perflux), which holds back the ion exchanger and allows the mycelium to pass through, filtered.
  • the ion exchanger should be chosen so that the density of the resin is significantly greater than that of the mycelium.
  • the bound antibiotics are stable for at least 2-3 months.
  • the ion exchange resin is known in a known manner with salt solutions, for. B. sodium chloride, sodium acetate or potassium chloride, wherein to increase the elution yield, which can be about 55-80%, optionally a water-miscible organic solvent, such as. As methanol, isopropanol or acetone, preferably at a concentration of 20-80%, is added.
  • salt solutions for. B. sodium chloride, sodium acetate or potassium chloride, wherein to increase the elution yield, which can be about 55-80%, optionally a water-miscible organic solvent, such as. As methanol, isopropanol or acetone, preferably at a concentration of 20-80%, is added.
  • the overall yield of the first purification step is thus about 50-75%, based on the activity in the fermentation solution.
  • the activity of the thienamycin and olivanic acid antibiotics can in the usual way by microbiological tests (agar diffusion test, turbidimetric test) with suitable test germs, e.g. B. Escherichia coli, Bacillus subtilis.
  • suitable test germs e.g. B. Escherichia coli, Bacillus subtilis.
  • the determination of the ⁇ -lactamase inhibitor effect eg. B. with the help of the new chromogenic ⁇ -lactamase substrates PADAC and CENTA (P. Schindler et al, Abstracts 21st Intersience Conference on Antimicrobial Agents and Chemotherapy, Chicago, 1981).
  • PADAC and CENTA P. Schindler et al, Abstracts 21st Intersience Conference on Antimicrobial Agents and Chemotherapy, Chicago, 1981.
  • the activity determined by these methods is described in Enzyme inhibitor units (UE 50 / ml) specified.
  • 10 l fermentation solution of the strain Streptomyces Y 5633A (DSM 2323), which forms several olivic acid components and, after fermentation (see, for example, Antibiot. 32, 295-304, 1979), the maximum activity after 42 hours, measured in enzyme Inhibitor units, with 15 units 50 / ml 150,000 units 50, are achieved by adding cold water while stirring and simultaneously cooling to 6 ° C. at this point with 300 g of Dowex ® 1 x 4, 20-50 anion exchange resin mesh, offset. After 30 minutes, the fermentation solution is poured into a funnel-shaped container with a bottom drain and the exchange resin is allowed to settle.
  • DSM 2323 strain Streptomyces Y 5633A
  • the resin drawn off after sufficient sedimentation over the soil is decanted to remove the mycelium residues washed with water and then in the usual manner, for. B. processed by elution.
  • the adsorbate obtained is treated with cooling and stirring with 1.2 l of a solution of 6% KCI in 50% acetone and the resin is separated off by filtration after 3 hours.
  • the adsorbate obtained in this way can be stored at - 20 ° C for at least 2 months without loss of activity.
  • the ion exchange resin is filled into a column with a cooling jacket and eluted with cooling (6 ° C.) with 6% potassium chloride in 50% acetone.
  • the 500 ml fractions are checked for activity in the inhibitor test.
  • the fractions are combined, concentrated to 0.9 I under high vacuum, and placed on a cooled column of 6 l of Diaison HP-20 adsorption resin, and the activity is eluted with cold water.
  • the crude antibiotic mixture obtained in this way is further purified in a known manner.
  • the streptomycete strain Y 5633 A (DSM 2323) producing olivic acids is cultivated in a 3 l nutrient solution in the customary manner and the fermentation solution after 38 hours when the maximum product formation has been reached to a suspension of 90 g of Amberlite® IRA-401 cooled to 4 ° C. S, 20-50 mesh, placed in 6 l of water and the resin separated in the manner described in Example 1.
  • the resin contains 90% of the activity measured in the fermentation solution.
  • the Amberlite IRA-401 S is filled into a column (2.5 x 30 cm) and the activity is eluted with a linear gradient of 50% acetone and 8% KCI in 50% acetone (fraction size 20 ml). The main activity is found in fractions 11-12, the elution yield is 55%.

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  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Nitrogen Condensed Heterocyclic Rings (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Claims (7)

1. Procédé d'isolement d'antibiotiques du Car- bapénème chimiquement instables de solutions de fermentation, caractérisé en ce que les antibiotiques formés sont adsorbés au maximum de la formation de produit sur des résines échangeuses d'anions appropriées, amenés par ce moyen sous une forme stabilisée et en ce que la résine est séparée de la solution de fermentation.
2. Procédé suivant la revendication 1, caractérisé en ce qu'on utilise comme résines échangeuses d'anions des résines ayant une basicité moyenne à forte.
3. Procédé suivant la revendication 2, caractérisé en ce que les résines échangeuses d'anions possèdent de préférence une granulométrie de 300 à 800 µm.
4. Procédé suivant la revendication 1, caractérisé en ce qu'on ajoute la résine échangeuse d'anions au moment approprié à la solution de fermentation, et en ce qu'on refroidit rapidement celle-ci.
5. Procédé suivant la revendication 1, caractérisé en ce qu'on pompe la solution de fermentation dans un récipient dans lequel se trouve la résine échangeuse d'anions, en suspension dans de l'eau froide.
6. Procédé suivant la revendication 1, caractérisé en ce qu'on sépare les échangeurs d'anions de la solution de fermentation par sédimentation.
7. Procédé suivant la revendication 1, caractérisé en ce qu'on sépare les échangeurs d'anions de la solution de fermentation par filtration sur un tamis approprié, qui retient l'échangeur et laisse passer le mycélium.
EP82110641A 1981-11-21 1982-11-18 Isolation d'antibiotiques chimiquement instables de solutions de fermentation Expired EP0080166B1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AT82110641T ATE15227T1 (de) 1981-11-21 1982-11-18 Isolierung von chemisch instabilen antibiotika aus fermentationsloesungen.

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE3146190 1981-11-21
DE19813146190 DE3146190A1 (de) 1981-11-21 1981-11-21 Isolierung von chemisch instabilen antibiotika aus fermentationsloesungen

Publications (2)

Publication Number Publication Date
EP0080166A1 EP0080166A1 (fr) 1983-06-01
EP0080166B1 true EP0080166B1 (fr) 1985-08-28

Family

ID=6146895

Family Applications (1)

Application Number Title Priority Date Filing Date
EP82110641A Expired EP0080166B1 (fr) 1981-11-21 1982-11-18 Isolation d'antibiotiques chimiquement instables de solutions de fermentation

Country Status (7)

Country Link
US (1) US4529545A (fr)
EP (1) EP0080166B1 (fr)
JP (1) JPS58101696A (fr)
AT (1) ATE15227T1 (fr)
CA (1) CA1199885A (fr)
DE (2) DE3146190A1 (fr)
ES (1) ES8307894A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SI9400107A (en) 1994-03-02 1995-10-31 Lek Tovarna Farmacevtskih New process of the isolation of clavulanic acid and its pharmaceutical salts from fermented broth of streptomyces sp.p 6621 ferm p 2804.

Family Cites Families (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3973015A (en) * 1970-08-03 1976-08-03 Eli Lilly And Company Antibiotic A16884
US4054564A (en) * 1970-08-03 1977-10-18 Eli Lilly And Company 7-(5-Amino-5-carboxyvaleramido)-7-methoxycephalosporanic acid
US3962427A (en) * 1972-12-08 1976-06-08 Eli Lilly And Company Antibiotic nebramycin factor VII
US4000161A (en) * 1974-12-19 1976-12-28 Merck & Co., Inc. Process for purifying thienamycin
US3993748A (en) * 1975-01-20 1976-11-23 The Upjohn Company Antibiotic U-47,929 and its preparation
DE2528622A1 (de) * 1975-06-26 1977-01-13 Bayer Ag Verfahren zur reinigung der bei der enzymatischen spaltung von beta-lactam- antibiotika anfallenden produkte
US4229534A (en) * 1975-11-21 1980-10-21 Merck & Co., Inc. Acetylthienamycin production
US4282322A (en) * 1975-11-24 1981-08-04 Merck & Co., Inc. Process for enzymatic deacylation of antibiotics
DK497476A (da) * 1975-11-24 1977-05-25 Merck & Co Inc Fremgangsmade til fremstilling af et antibiotisk stof
SE7801695L (sv) * 1977-03-01 1978-09-02 Merck & Co Inc Tienamycin
GR62185B (en) * 1977-03-31 1979-03-02 Panlabs Inc Preparation process of an antibiotic ps-5 and its derivatives with inhibitory activity of b-lactamase
GB1583841A (en) * 1977-07-14 1981-02-04 Beecham Group Ltd Antibiotics mm4550 mm13902 and mm17880 preparation
JPS55136282A (en) * 1979-04-06 1980-10-23 Shionogi & Co Ltd Novel antibiotic pa-31088-4
US4247640A (en) * 1979-07-23 1981-01-27 Merck & Co., Inc. Fermentation process for 6-hydroxymethyl-2-(2-aminoethylthio)-1-carbadethiapen-2-em-3-carboxylic acid
EP0024447B1 (fr) * 1979-08-28 1983-07-20 Merck & Co. Inc. Procédé de purification de la thiénamycine
KR840001256B1 (ko) * 1980-08-19 1984-09-01 메이지세카 주식회사 신항생물질 sf-2103a 물질의 제조법

Also Published As

Publication number Publication date
DE3265895D1 (en) 1985-10-03
ES517485A0 (es) 1983-08-01
US4529545A (en) 1985-07-16
ES8307894A1 (es) 1983-08-01
JPS58101696A (ja) 1983-06-16
DE3146190A1 (de) 1983-06-16
EP0080166A1 (fr) 1983-06-01
CA1199885A (fr) 1986-01-28
ATE15227T1 (de) 1985-09-15

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